PROJECT SUMMARY/ABSTRACT Hemophilia A affects 20,000 people in the US, classifying it as an Orphan Disease, and is characterized by the inability to produce functional factor VIII (fVIII) ? a plasma protein necessary for blood clot formation. 30% of those with severe hemophilia A have inhibitory antibodies to the commonly used drug, which is intravenous infusion of plasma-derived or recombinant fVIII. These patients represent a severely underserved population, as they are left with only two on-demand treatment options for bleeding episodes that either require multiple infusions or long infusion times. Further complicating treatment, potentially fatal thrombotic side effects have been seen when newly approved prophylactic therapies are combined with on-demand therapies. Moreover, it is not uncommon for these patients to spend upwards of $1,000,000 per year on their treatment therapy alone. To address this severe patient need, Split Rock Therapeutics has developed a polyelectrolyte multilayer (polymer) microcapsule for the targeted delivery of fVIII for hemophilia patients with anti-fVIII inhibitors. FVIII is encapsulated within the polymer shell, and, upon intravenous administration, binds to and hybridizes with the patient?s native platelets. The hybridized platelets then target the capsule to sites of injury and rupture the capsule open through their natural contractile behavior in the clot formation process to facilitate a ?burst? release of fVIII. Importantly for patients with inhibitors, the fVIII is protected from the patient?s inhibitors during circulation by the polymer shell, so a near-full dose of uninhibited fVIII is delivered at the site of injury to quickly rescue hemostasis. Our recent publication (Hansen, C. E. et al. ACS Nano 2016) describes in vitro investigations into the platelet- mediated mechanism of the targeted drug delivery technology, leveraging the expertise in platelet contraction behavior of Dr. Wilbur A. Lam (Lam, W. et al. Nature Mater. 2011; Qiu, Y. et al. PNAS 2014; Myers, D. et al. Nature Mater. 2017). Proof-of-concept in vivo investigations demonstrate safety and significantly enhanced efficacy of the fVIII loaded microcapsules compared to infusion of fVIII alone. For this Phase I SBIR, Split Rock Therapeutics proposes investigations into pharmacological parameters of this technology to determine dosages that are safe and efficacious. In Milestone 1, we propose to investigate plasma concentrations over time, organ biodistribution, and the kinetics of platelet-capsule hybridization. These studies will result in dose-exposure data that will enable the determination of key pharmacokinetic parameters required for effective dosing in Milestone 2. Dose-response relationships will be determined for both hemostatic and adverse responses in Milestone 2. This pharmacodynamic data will enable calculation of dosages that are efficacious and exhibit a low risk of side effects. This proposal will facilitate a partnership with pharmaceutical companies in order to conduct further investigations with an already FDA approved fVIII drug. Future areas of investigation include metabolism and elimination of the therapy, additional injury models of varying severity, and investigation into non-specific safety biomarkers.